Molecular determinants of myeloma bone disease and uses thereof

ABSTRACT

To identify molecular determinants of lytic bone disease in multiple myeloma, the expression profiles of ˜12,000 genes in CD138-enriched plasma cells from newly diagnosed multiple myeloma patients exhibiting no radiological evidence of lytic lesions (n=28) were compared to those with ≧3 lytic lesions (n=47). Two secreted WNT signaling antagonists, soluble frizzled related protein 3 (SFRP-3/FRZB) and the human homologue of Dickkopf-1 (DKK1), were expressed in 40 of 47 with lytic bone lesions, but only 16 of 28 lacking bone lesions (P&lt;0.05). DKK1 and FRZB were not expressed in plasma cells from 45 normal bone marrow donors or 10 Waldenstrom&#39;s macroglobulinemia, a related plasma cells malignancy that lacks bone disease. These data indicate that these factors are important mediators of multiple myeloma bone disease, and inhibitors of these proteins may be used to block bone disease.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit of provisional patent application U.S.Ser. No. 60/431,040, filed Dec. 5, 2002, now abandoned.

FEDERAL FUNDING LEGEND

This invention was created, in part, using funds from the federalgovernment under National Cancer Institute grants CA55819 and CA97513.Consequently, the U.S. government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the study of multiplemyeloma. More specifically, the present invention relates to theidentification of molecular determinants of myeloma bone disease throughcomparative global gene expression profiling.

2. Description of the Related Art

Multiple myeloma (MM) is a rare, yet incurable malignancy of terminallydifferentiated plasma cells (PC) that affects approximately 15,000persons per year in the United States, and represents the second mostcommon hematopoietic malignancy. Multiple myeloma represents 13% of alllymphoid malignancies in the white population and 31% of lymphoidmalignancies in the black population. The malignant plasma cells home toand expand in the bone marrow causing anemia and immunosuppression dueto loss of normal hematopoiesis.

Multiple myeloma is also associated with systemic oteoporosis and localbone destruction leading to debilitating bone pain and susceptibility tofractures, spinal cord compression and hypercalcemia. Myeloma is theonly hematological malignancy consistently associated with lytic bonedisease and local bone destruction is limited to areas adjacent toplasma cells, suggesting that the malignant plasma cells secrete factorsthat enhance osteoclast function and/or osteoblast anergy. Theprevalence of bone disease varies with the presentation of myeloma, fromsmoldering myeloma, often without bone involvement, to solitaryplasmacytoma, to diffused or focal multiple myeloma where systemiclosses of bone mineral density or focal lytic bone lesions are seen inapproximately 80% of patients.

In recent years, it has become evident that lytic bone disease is notonly a consequence of myeloma, but that it is intricately involved inpromoting disease progression. Change in bone turnover rates predictsclinical progression from monoclonal gammopathy of undeterminedsignificance (MGUS) to overt myeloma by up to 3 years. While initiallyosteoclast and osteoblast activity are coupled, the coupling is lostwith disease progression. Osteoclast activity remains increased andosteoblast activity is diminished, with lytic bone disease as theconsequence. Studies in the 5T2 murine myeloma and the SCID-hu model forprimary human myeloma demonstrated that inhibition of osteoclastactivity is associated with inhibition of myeloma growth and reductionof myeloma tumor burden. These studies support reports that inhibitionof bone resorption with bisphosphonates had an anti-myeloma effect.

Whereas the biology of osteoclasts in myeloma-associated lytic bonedisease has been investigated intensively, little is known about thedisease-associated changes in osteoblast activity and their underlyingmechanisms. It has been suggested that in myeloma, the ability ofmesenchymal stem cells to differentiate into the osteogenic lineage isimpaired. However, the mechanisms responsible for such impairment havenot been elucidated.

It has been shown that comparative global gene expression profiling(GEP) of bone marrow plasma cells from normal healthy donors andmalignant bone marrow plasma cells from newly diagnosed multiple myelomarepresented a powerful technique for identifying candidate disease genesand disrupted pathways involved in malignant transformation of multiplemyeloma (Zhan et al., 2002).

The prior art is deficient in a comparative analysis to identify genesexpressed in the malignant plasma cells that may be contributory tomultiple myeloma bone diseases as well as methods to diagnose and treatmultiple myeloma bone diseases. The present invention fulfills thislongstanding need and desire in the art.

SUMMARY OF THE INVENTION

To identify the molecular determinants of lytic bone disease, theexpression profiles of ˜12,000 genes in CD138-enriched plasma cells fromnewly diagnosed multiple myeloma exhibiting no radiological evidence oflytic lesions (n=28) were compared to those with ≧3 lytic lesions(n=47). Consistent with a critical role of WNT signaling in osteoblastdifferentiation, two secreted WNT signaling antagonists, solublefrizzled related protein 3 (SFRP-3/FRZB) and the human homologue ofDickkopf-1 (DKK-1), were expressed in 40 of 47 with lytic bone lesions,but only 16 of 28 lacking bone lesions (P<0.05). Immunohistochemistryshowed high levels of DKK-1 and FRZB in plasma cells from cases withhigh gene expression. Importantly, DKK-1 and FRZB were not expressed inplasma cells from 45 normal bone marrow donors or 10 Waldenstrom'smacroglobulinemia, a related plasma cells malignancy that lacks bonedisease.

Serum derived from multiple myeloma patients with high DKK-1 blockedboth Wnt signaling and osteoblast differentiation in vitro. Importantly,pre-incubation of the serum with DKK-1 and FRZB antibodies inhibitedthis function. Consistent with a key role for JUN in controlling DKK-1expression and in turn apoptosis, plasma cells derived fromextramedullary disease as well as primary refractory disease had lowexpression of JUN and DKK-1.

Multiple myeloma plasma cells showed a massive up-regulation of DKK-1and FRZB gene expression after in vivo treatment. DKK-1 and FRZB can beupregulated in multiple myeloma plasma cells after treatment of patientswith genotoxic drugs used to treat the disease, thus furthering a rolefor DKK-1 in multiple myeloma cell apoptosis. Primary multiple myelomacells co-cultured with in vitro derived osteoclasts (OC) lackedapoptosis and that this was tightly correlated with the down-regulationof JUN, FOS, FOSB, and DKK-1.

Results disclosed in the present invention indicate that blocking theproduction and/or secretion of DKK-1 and FRZB may prevent or reversebone loss in multiple myeloma patients. Further applications may includeusing DKK-1 and FRZB inhibitors to prevent bone loss in the generalpopulation. Additionally, Wnt signaling has recently been shown to becritical for the self renewal capacity of hematopoietic stem cells.Futhermore, a bone marrow niche required for HSC proliferation is formedby mature osteoblasts. The block to Wnt signaling by DKK1 and FRZB coulddirectly and indirectly impair hepatic stellate cell (HSC) proliferationand thus may partly account for the immunosuppression and anemia seen inmultiple myeloma. Thus blocking DKK1 and/or FRZB may also prevent orreverse the defect in hematopoeisis seen in most patients with myeloma.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention as well as others which will become clear areattained and can be understood in detail, more particular descriptionsand certain embodiments of the invention briefly summarized above areillustrated in the appended drawings. These drawings form a part of thespecification. It is to be noted, however, that the appended drawingsillustrate preferred embodiments of the invention and therefore are notto be considered limiting in their scope.

FIGS. 1A and 1B show global gene expression patterns reflecting bonelesions in myeloma. FIG. 1A shows clusterview of normalized expressionlevels of 57 genes identified by logistic regression analysis as beingsignificantly differentially expressed in malignant plasma cells frompatients with no (n=36) and 1+MRI focal lesions (n=137) (P<0.0001). The28 genes exhibiting elevated expression in plasma cells from patientswith 1+MRI lesions are ordered from top to bottom based on rank ofsignificance. Likewise the 30 genes showing significant elevation inpatients with no MRI-lesions are ordered from bottom to top based onsignificance rank. Gene symbols (Affymetrix probe set identifiers whenthe gene is unnamed) are listed to the left. Normalized expressionscales range from −30 (blue) to +30 (red) as indicated below the datadisplay. The four genes remaining significant after permutationadjustment are underlined.

FIG. 1B shows a bar graph of DKK1 gene expression in plasma cells fromnormal bone marrow (BPC), patients with monoclonal gammopathy ofundetermined significance (MGUS), Waldenström's macroglobulinemia (WM),and multiple myeloma (MM) presented on the x-axis. MM samples are brokendown into three bone lesion groups: no MRI/no x-ray lesions, 1+MRI/nox-ray lesions, and 1+MRI/1+x-ray lesions. The Affymetrix Signal, aquantitative measure of gene expression derived from MAS 5.01, isindicated on the y-axis. DKK1 gene expression level in each sample isindicated by a bar, with the height of the bar proportional to geneexpression intensity. Samples are ordered from the lowest to highestDKK1 gene expression from left to right on the x-axis. The number ofsamples in each group is indicated below each group designator.Statistics for comparisons between the MM subgroups are indicated in thetext.

FIG. 2 shows RHAMM was up-regulated in multiple myeloma patients withbone lesions.

FIG. 3 shows RHAMM rarely present in normal plasma cells and monoclonalgammopathy of undetermined significance (MGUS), but it was present invirtually all human myeloma cell lines.

FIG. 4 shows securin was up-regulated in multiple myeloma patients withbone disease.

FIG. 5 shows MIP-1α and CCR1 were “spike” genes in multiple myeloma, butthey were not correlated with lytic lesions. Black bar: CCR1; gray bar:MIP-1α.

FIG. 6 shows MIP-1α was expressed at low level in normal plasma cells(PC).

FIG. 7 shows the expression of WNT antagonist DKK-1 in multiple myelomawith bone lesions.

FIG. 8 shows the expression of WNT decoy receptor FRZB in multiplemyeloma with lytic bone lesions.

FIG. 9 shows the expression of DKK-1 and FRZB in multiple myeloma withlytic bone lesions. Black bar: DKK-1; gray bar: FRZB.

FIG. 10 shows FRZB was expressed in tonsil plasma cells. PBC, TBC,tonsil B cells; TPC, tonsil plasma cells; BPC, bone marrow plasma cells;WPC, WBC, CLL.

FIG. 11 shows DKK-1 was not expressed in normal B cells or plasma cells.PBC, TBC, tonsil B cells; TPC, tonsil plasma cells; BPC, bone marrowplasma cells; WPC, WBC, CLL.

FIG. 12 shows DKK-1 expression in monoclonal gammopathy of undeterminedsignificance (MGUS) was low relative to smoldering multiple myeloma(SMM) and newly diagnosed multiple myeloma (MM).

FIG. 13 shows FRZB was elevated in monoclonal gammopathy of undeterminedsignificance (MGUS), and had higher expression in smoldering multiplemyeloma (SMM) and newly diagnosed multiple myeloma (MM).

FIG. 14 shows the expression of DKK-1 and FRZB in monoclonal gammopathyof undetermined significance (MGUS) and smoldering multiple myeloma(SMM).

FIG. 15 shows low expression of DKK-1 in extramedullary disease.

FIG. 16 shows the expression of DKK-1 and FRZB tend to be higher inplasma cells from medullary PCT than those from iliac crest. PCT, FNA.

FIG. 17 shows the expression of DKK-1 and FRZB in fine needle aspiratesof medullary PCT.

FIG. 18 shows high expression of DKK-1 and FRZB in medullaryplasmacytoma.

FIG. 19 shows higher expression of DKK-1 in multiple myeloma withosteopenia.

FIG. 20 shows DKK-1 was not expressed in plasma cells from Waldenstrom'smacroglobulinemia.

FIG. 21 shows WNT5A was elevated in newly diagnosed multiple myeloma.

FIG. 22 shows WNT5A tends to be higher in multiple myeloma with lyticlesions.

FIG. 23 shows WNT5A was also elevated in monoclonal gammopathy ofundetermined significance (MGUS) and smoldering multiple myeloma (SMM).

FIG. 24 shows WNT10B tends to be lower in multiple myeloma with lyticlesions.

FIG. 25 shows WNT5A and WNT10B tend to be inversely correlated. Blackbar: WNT10B; gray bar: WNT5A.

FIG. 26 shows DKK-1 was present in an SK-LMS cell line.

FIG. 27 shows primary multiple myeloma synthesized DKK-1 protein.

FIG. 28 shows low DKK-1 expression in relapsed and primary refractorymultiple myeloma.

FIG. 29 shows endothelin receptor B was a “spike” gene in one third ofnewly diagnosed multiple myeloma.

FIG. 30 shows the expression of endothelin receptor B in monoclonalgammopathy of undetermined significance (MGUS) and smoldering multiplemyeloma. Normal plasma cells do not express endothelin receptor B.

FIG. 31 shows the involvement of endothelin receptor B in boneformation.

FIG. 32 shows DKK-1 expression after treatment with PS-341.

FIG. 33 shows DKK-1 expression after treatment with thalomid in newlydiagnosed multiple myeloma.

FIG. 34 shows DKK-1 expression after treatment with IMiD.

FIG. 35 shows DKK-1 expression after treatment with dexamethsone innewly diagnosed multiple myeloma.

FIG. 36 shows downregulation of JUN and FOS in multiple myeloma cellsafter co-culture with osteoclasts.

FIG. 37 shows JUN & DKK-1 downregulation in osteoclast co-culture.

FIG. 38 shows WNT signaling in multiple myeloma bone disease.

FIG. 39 shows overexpression of DKK1 in low grade myeloma with the lossof expression with disease progression. Expression of DKK1 was examinedby immunohistochemistry of myeloma bone marrow biopsies. Serial sections(550× magnification) of bone marrow biopsies from myeloma patients withhigh (a-b) and low (c-d) DKK1 gene expression are presented. Slides arestained with H&E (a and c) or anti-DKK1 and secondary antibody (b andd). Use of secondary alone failed to stained cells (data not shown).Magnified images (1,200× magnification) are located in the upper leftcorner of each H&E image. Image a shows a myeloma with an interstitialpattern of involvement with plasma cells exhibiting low grade morphologywith abundant cytoplasm and no apparent nucleoli. Image b revealspositive staining of plasma cells in a interstitial pattern withanti-DKK1 antibody that was greatest adjacent to bone. Image c shows amyeloma with nodular or alliterative pattern with plasma cellsexhibiting high grade morphology with enlarged nuclei and prominentnucleoli. Image d reveals no positive staining of plasma with anti-DKK1antibody.

FIGS. 40A and 40B show DKK1 protein in the bone marrow plasma is highlycorrelated with DKK1 gene expression and the presence of bone lesions.FIG. 40A shows the expression of DKK1 mRNA was detected by microarrayand DKK1 protein by ELISA in a total of 107 cases of newly diagnosedmyeloma. Results of both assays were transformed by the log base 2 andnormalized to give a mean of 0 and variance of 1. Each bar indicates therelative relationship of gene expression and protein expression in eachsample. There was a significant correlation between DKK1 mRNA in myelomaplasma cells and protein in bone marrow plasma (r=0.65, P<0.001). FIG.40B shows bar view of DKK1 protein levels in bone marrow plasma plasmacells from normal donors (BPC), patients with monoclonal gammopathy ofundetermined significance (MGUS), Waldenström's macroglobulinemia (WM),and multiple myeloma (MM) are presented on the x-axis. MM samples arebroken down into three bone lesion groups: no MRI/no x-ray lesions,1+MRI/no x-ray lesions, and 1+MRI/1+x-ray lesions. The DKK1 proteinconcentration (ng/ml) is indicated on the y-axis. To enable comparisonsof DKK1 protein levels in the lower ranges, 200 ng/ml was made themaximum value. This resulted in the truncation of a single sample withDKK1 concentration of 476 ng/ml. DKK1 protein level in each sample isindicated by a bar, with the height of the bar proportional to DKK1protein levels. Samples are ordered from the lowest to highest DKK1protein levels from left to right on the x-axis. The number of samplesin each group is indicated below each group.

FIGS. 41A and 41B show recombinant DKK1 and MM plasma can block alkalinephosphatase production in BMP-2 treated C2C12 cells in a DKK1-dependentmanner. FIG. 41A shows alkaline phosphatase levels, a marker ofosteoblast differentiation (y-axis) were measured in C2C12 cells after 5days of culture in the presence of 5 percent fetal calf serum alone orwith BMP2, BMP2+DKK1, BMP2+DKK1+anti-DKK1, or BMP-2+DKK1+polyclonal IgG.Each bar represents the mean (±SEM) of triplicate experiments. Note thatactivity of alkaline phosphatase increased in the presence of BMP-2 andsignificant reduction of this protein by co-incubation with recombinantDKK1. Also note that anti-DKK1 antibody, but not polyclonal IgG canblock the repressive activity of DKK1. FIG. 41B shows alkalinephosphatase levels (y-axis) were tested in C2C12 cells after culturingthese cells for 5 days in 5 percent fetal calf serum alone or 50 ng/mlBMP-2+10 percent normal bone marrow plasma (NS) or BMP-2+10 percentmyeloma bone marrow plasma from 10 patients with newly diagnosed myeloma(sample identified provided), or BMP2+10 percent myeloma patient plasma+anti-DKK1 or goat polyclonal IgG. Each bar represents the mean (±SEM)of triplicate experiments. DKK1 concentration from each bone marrowplasma samples was determined by ELISA and final concentrations inculture after 1:10 dilution are indicated on the x-axis. Note thatsamples with >12 ng/ml DKK1 had an effect on alkaline phosphataseproduction. A star indicates P<0.05 in comparison to alkalinephosphatase in BMP2+10 percent normal human bone marrow plasma.

DETAILED DESCRIPTION OF THE INVENTION

The present invention demonstrates that the secreted WNT signalingantagonists DKK-1 and FRZB mediate bone destruction seen in multiplemyeloma. Together with emerging evidence of an absolute requirement ofWnt signaling in osteoblast growth and differentiation, these datastrongly implicate these factors in causing osteoblast anergy andcontributing to multiple myeloma bone disease by suppressing the normalcompensatory bone production that follows bone loss.

The role of multiple myeloma plasma cells in stimulating osteoclastactivity has been intensely investigated and several key linksestablished. Data presented herein provide for the first time evidenceof a possible mechanistic explanation of osteoblast dysfunction inmultiple myeloma. These are significant observations in that recentstudies have shown that inhibition of WNT signaling causes defects inosteoblast function. The secreted DKK-1 and FRZB could account for boththe systemic osteoporosis seen in multiple myeloma as well as theexaggerated local bone destruction proximal to plasma cells foci.

Importantly, DKK-1 and FRZB act to inhibit WNT signaling throughindependent mechanisms, indicating that their co-expression may havesynergistic effects. Thus, these genes could be used to predict extentof bone disease and future risk of developing bone disease. Moreover,inhibitors of these proteins could be used to block bone disease. It isalso possible that these factors play a role in osteoporosis in thegeneral population.

WNT Signaling Pathway

Wnt genes comprise a large family of secreted polypeptides that areexpressed in spatially and tissue-restricted patterns during vertebrateembryonic development. Mutational analysis in mice has shown theimportance of Wnts in controlling diverse developmental processes suchas patterning of the body axis, central nervous system and limbs, andthe regulation of inductive events during organogenesis. The Wnt familyof secreted growth factors initiates signaling via the Frizzled (Fz)receptor and its coreceptor, LDL receptor-related protein 5 or 6 (LPR5or LRP6), presumably through Fz-LPR5/LRP6 complex formation induced byWnt.

Secreted antagonists of Wnt include Frizzled (Fz)-related proteins(FRPs), Cerberus, Wnt inhibitory factor (WIF) and Dickkopf (DKK).Frizzled (Fz)-related proteins, Cerberus and Wnt inhibitory factor haveall been shown to act by binding and sequestering Wnt. Unlike Wntantagonists which exert their effects by molecular mimicry of Fz or Wntsequestration through other mechanisms, Dickkopf-1 (DKK-1) specificallyinhibits canonical Wnt signalling by binding to the LPR5/LRP6 componentof the receptor complex.

DKK-1 is a head inducer secreted from the vertebrate head organizer andinduces anterior development by antagonizing Wnt signaling. DKK-1 is ahigh-affinity ligand for LRP6 and inhibits Wnt signaling by preventingFz-LRP6 complex formation induced by Wnt. DKK-1 binds neither Wnt norFz, nor does it affect Wnt-Fz interaction. DKK-1 function in headinduction and Wnt signaling inhibition strictly correlates with itsability to bind LPR5/LRP6 and to disrupt the Fz-LPR5/LRP6 association.LPR5/LRP6 function and DKK-1 inhibition appear to be specific for theWnt/Fz beta-catenin pathway. These findings thus reveal a novelmechanism for Wnt signal modulation.

WNT Signaling and Osteoblast Differentiation

Recent studies have shown that the Wnt signaling pathway is critical forosteoblast differentiation and function. Mice with a targeted disruptionin the gene for low-density lipoprotein receptor-related protein 5(LRP5) developed a low bone mass phenotype. LRP5 is expressed inosteoblasts and is required for optimal Wnt signaling in osteoblasts. Invivo and in vitro analyses indicated that this phenotype becomes evidentpostnatally, and it was secondary to decreased osteoblast proliferationand function in a Cbfa1-independent manner.

In human, mutations in LRP5 cause the autosomal recessive disorderosteoporosis-pseudoglioma syndrome (OPPG). Osteoporosis-pseudogliomasyndrome carriers have reduced bone mass when compared to age- andgender-matched controls.

Importantly, separate and distinct mutations in LRP result in a highbone mass phenotype. In contrast to the osteopororsis-psuedogliomamutations, the high bone mass traits are gain of function mutations.Markers of bone resorption were normal in the affected subjects, whereasmarkers of bone formation such as osteocalcin were markedly elevated.Levels of fibronectin, a known target of signaling by Wnt, were alsoelevated. In vitro studies showed that the normal inhibition of Wntsignaling by Dickkopf-1 (DKK-1) was defective in the presence of themutation and that this resulted in increased signaling due to unopposedWnt activity. These findings demonstrated the role of altered LRP5function in high bone mass and point to DKK as a potential target forthe prevention or treatment of osteoporosis.

WNT Signaling and Bone Disease in Multiple Myeloma

Indirect evidence of a role of DKK-1 in osteoblast function has beenprovided by identification of gain of function mutations in LRP-5 beinglinked to a high bone mass phenotype. In addition, targeted disruptionof secreted firzzled-related protein (SFRP-1), a homologue of FRZB(SFRP-3), leads to decreased osteoblast and osteocyte apoptosis andincreased trabecular bone formation.

A quantitative trait loci (QTL) influencing bone mass has been localizedto the LRP-5 region, suggesting that the population at large havedifferent risk of developing osteoporosis. It is conceivable thatmultiple myeloma bone disease may be influenced by the combined effectsof DKK-1/FRZB expression with an inherited predisposition to low bonemass conferred by inherited LRP-5 alleles. Multiple myeloma cases may begenotyped for LRP-5 allele variations and correlate this informationwith bone disease, and DKK-1 and FRZB expression.

Monoclonal gammopathy of undetermined significance (MGUS), a plasma celldyscrasia that is predisposed to develop into multiple myeloma, isdifferentiated from multiple myeloma by the lack of obvious bonedisease. The significance of discovering DKK-1 and/or FRZB expression ina third of monoclonal gammopathy of undetermined significance is unclearbut could suggest that these cases may be at higher risk for developingmultiple myeloma. As with multiple myeloma, this predisposition may alsobe related to inherited LRP5 alleles. Alternatively, these monoclonalgammopathy of undetermined significance cases could have underlyingpreclinical bone disease that is not yet apparent by radiological scans.

Data presented herein suggests a model for how DKK-1 expression bymultiple myeloma plasma cells can be linked to multiple myeloma diseasegrowth control and bone destruction and how these two phenomena can beintegrated by one molecule. In the model, primary multiple myelomaexpress high levels of DKK and these levels can be increased with drugtherapies used to treat the disease. High levels of DKK-1 likely induceapoptosis of multiple myeloma cells and could explain the relativelyslow progression of the disease in its early phase as cell growth istempered by high rate of DKK-1 induced apoptosis. However, as thedisease progresses there is an osteoclast-induced reduction in JUN andDKK-1 that eventually develops into a constitutive loss of JUN and DKK-1expression as seen in extramedullary disease.

Thus, if one were to view DKK-1 expression from the perspective of themultiple myeloma plasma cells, high levels of DKK-1 expression could beseen as positive feature of the disease. However, with the mesenchymalcell lineage being exquisitely sensitive to DKK-1 induced apoptosis, thehigh levels of this secreted product likely has a double edge to it inthat it also induces massive programmed cell death of osteoblastprecursors and possibly even mesenchymal stem cells. It is expected thathigh levels of DKK-1 early in the disease could lead to a permanent lossof mesenchymal stem cells, a notion supported by the observed lack ofbone repair after remission induction or during disease progression whenosteoclasts likely suppress DKK-1 secretion by multiple myeloma plasmacells. Thus, exploitation of this knowledge might lead to thedevelopment of new therapies for multiple myeloma that accentuateDKK-1's effects on multiple myeloma plasma cells, but at the same timeprevent DKK's bone damaging effects on osteoblast or their precursors.

In one embodiment of the present invention, there is provided a methodof determining the potential of developing a bone disease in a multiplemyeloma patient by examining the expression level of WNT siganlingantagonist. Increased expression of the antagonist compared to that innormal individual would indicate that the patient has the potential ofdeveloping bone disease. Preferably, the WNT signaling antagonist issoluble frizzled related protein 3 (SFRP-3/FRZB) or the human homologueof Dickkopf-1 (DKK1). In general, the expression levels of theseproteins can be determined at the nucleic acid or protein level.

In another embodiment, there is provided a method of treating bonedisease in a multiple myeloma patient by inhibiting the expression ofWNT signaling antagonist. Preferably, the WNT signaling antagonist issoluble frizzled related protein 3 (SFRP-3/FRZB) or the human homologueof Dickkopf-1 (DKK1). In general, the expression of these antagonistscan be inhibited at the nucleic acid or protein level.

In yet another embodiment, there is provided a method of preventing boneloss in an individual by inhibiting the expression of WNT signalingantagonist. Preferably, the WNT signaling antagonist is soluble frizzledrelated protein 3 (SFRP-3/FRZB) or the human homologue of Dickkopf-1(DKK1). In general, the expression of these antagonists can be inhibitedat the nucleic acid or protein level.

In yet another embodiment, there is provided a method of controllingbone loss in an individual, comprising the step of inhibiting theexpression of the DKK1 gene (accession number NM012242) or the activityof the protein expressed by the DKK1 gene. The DKK1 gene expression isinhibited by any method known to a person having ordinary skill in thisart including, e.g., anti-sense oligonucleotides or by anti-DKK1antibodies or soluble LRP receptors.

In yet another embodiment, there is provided a method of controllingbone loss in an individual, comprising the step of administering to saidindividual a pharmacological inhibitor of DKK1 protein. Generally, thismethod would be useful where the individual has a disease such asmultiple myeloma, osteoporosis, post-menopausal osteoporosis ormalignancy-related bone loss. Generally, the malignancy-related boneloss is caused by breast cancer metastasis to the bone or prostatecancer metastasis to the bone.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

EXAMPLE 1

Patients

174 patients with newly diagnosed multiple myeloma, 16 patients withmonoclonal gammopathy of undetermined significance, 9 with Waldenström'smacroglobulinemia, and 45 normal persons were studied. The InstitutionalReview Board of the University of Arkansas for Medical Sciences approvedthe research studies and all subjects provided written informed consent.Table 1 shows the characteristics of the patients with multiple myeloma.

TABLE 1 Myeloma patient characteristics and their relationship to MRIlesions P Variable n/N % MRI = 1+ MRI = 0 value Age ≧ 65 yr  23/169 14 17/132  6/36 0.59* (12.9%) (16.7%) Caucasian 147/169 87 113/132 33/360.42* (85.6%) (91.7%) Female  68/169 40  55/132 13/36 0.55 (41.7%)(36.1%) Kappa light 104/165 63  79/128 24/36 0.59 chain (61.7%) (66.7%)Lambda light  61/165 37  49/128 12/36 0.59 chain (38.3%) (33.3%) IgAsubtype  39/169 23  25/132 14/36 0.012 (18.9%) (38.9%) B2M ≧ 4 mg/L 60/169 36  47/132 13/36 0.96 (35.6%) (36.1%) CRP ≧ 4 mg/L  12/166  7 11/129 (8.5%)  1/36 (2.8%) 0.47* Creatinine ≧  19/169 11  16/132  3/36(8.3%) 0.77* 2 mg/dL (12.1%) LDH ≧ 190 UI/L  52/169 31  44/132  8/360.20 (33.3%) (22.2%) Albumin < 3.5 g/dL  23/169 14  19/132  4/36 0.79*(14.4%) (11.1%) Hgb < 10 g/dL  40/169 24  31/132  8/36 0.87 (23.5%)(22.2%) PCLl ≧ 1%  23/150 15  18/119  4/30 1.00* (15.1%) (13.3%) ASPC ≧33% 109/166 66  82/129 26/36 0.33 (63.6%) (72.2%) BMPC ≧ 33% 104/166 63 79/129 24/36 0.55 (61.2%) (66.7%) Cytogenetic  52/156 33  45/121  6/340.032 abnormalities (37.2%) (17.6%) CA13 or  33/52 63  31/121  3/34(8.8%) 0.037 hypodiploid (25.6%) Other CA  19/52 37  53/103 16/32 0.89(51.5%) (50.0%) FISH13  69/136 51 103/136 28/36 0.80 (75.7%) (77.8%)Osteopenia 131/173 76 1+ Lesions 137/173 79 by MRI 3+ Lesions 108/173 62by MRI 1+ Lesions 105/174 60 by X-ray 3+ Lesions  69/174 40 by X-ray*Fisher's Exact test, otherwise Chi-square test

EXAMPLE 2

Bone Imaging

Images were reviewed, without prior knowledge of gene expression data,using a Canon PACS (Picture Archiving and Cataloging System). MRI scanswere performed on 1.5 Tesla GE Signa™ scanners. X-rays were digitizedfrom film in accordance with American College of Radiology standards.MRI scans and x-rays were linked to the Canon PACS system using theACR's DICOM (Digital Imaging and Communications in Medicine) standard.Imaging was done in accordance with manufacturers' specifications. MRIimages were created with pre- and post-gadolinium T1-weighting and STIR(short-tau inversion recovery) weighting.

EXAMPLE 3

Plasma Cell Isolation and Gene Expression Profiling

Following Ficoll-Hypaque gradient centrifugation, plasma cells obtainedfrom the bone marrow were isolated from the mononuclear cell fraction byimmunomagnetic bead selection using a monoclonal mouse anti-human CD138antibody (Miltenyi-Biotec, Auburn, Calif.). More than 90 percent of thecells used for gene expression profiling were plasma cells, as shown bytwo-color flow cytometry using CD138⁺/CD45⁻ and CD38⁺/CD45⁻ markers, thepresence of cytoplasmic immunoglobulin light chains byimmunocytochemistry, and morphology by Wright-Giemsa staining. Total RNAwas isolated with RNeasy Mini Kit (Qiagen, Valencia, Calif.).Preparation of labeled cRNA and hybridization to U95Av2 microarrayscontaining approximately 10,000 genes (Affymetrix, Santa Clara, Calif.)was performed as previously described (Zhan et al., 2002; Zhan et al.,2003). RNA amplification was not required.

EXAMPLE 4

Immunohistochemistry

An antibody from a goat that was immunized against the entire human DKK1protein (R&D Systems, Minneapolis, Minn.) was diluted 1:200 inTris-buffer and added to formalin-fixed, paraffin-embedded bone marrowbiopsy sections for 2 hours at room temperature. Adjacent sections werestained with H & E. Antigen-antibody reactions were developed with DAB(after biotinylated anti-goat antibody [Vector Laboratories, Burlingame,Calif.] [1:400 dilution] and streptavidin-horse radish peroxidase [Dako]staining), and counterstained with Hematoxylin-2.

EXAMPLE 5

Enzyme Linked Immunosorbent Assay (ELISA)

Nunc-Immuno MaxiSorp surface microtiter plates were coated with 50 ml ofanti-DKK1 antibody at 1 mg/ml in 1× phosphate buffered saline, pH 7.2 at4° C. overnight, and blocked with 4 percent bovine serum albumin. Bonemarrow plasma was diluted 1:50 in dilution buffer (1× phosphate bufferedsaline +0.1 Tween-20+1 percent bovine serum albumin). A total of 50 μlwas loaded per well and incubated overnight at 4° C., washed andincubated with biotinylated goat anti-human DKK1 IgG (R&D Systems)diluted to 0.2 mg/ml in dilution buffer, followed by addition of 50 μlof 1:10,000 dilution of streptavidin-horse radish peroxidase (VectorLaboratories), all according to manufacturer's recommendations. Colordevelopment was achieved with the OPD substrate system (Dako) based onmanufacturer's instructions. Serial dilutions of recombinant human DKK1(R&D Systems) were used to establish a standard curve. The cell lineT293, which does not express endogenous DKK1 and T293 with stablytransfected DKK1 (Fedi, et al., 1999) were used to validate the ELISAassay.

EXAMPLE 6

Osteoblast Differentiation Assays

C2C12 mesenchymal precursor cells (American Type Tissue Culture, Reston,Va.) were cultured in DMEM (Invitrogen, Carlsbad, Calif.) supplementedwith 10 percent heat-inactivated fetal calf serum. Alkaline phosphataseactivity in C2C12 cells was measured as described (Gallea, et al., 2001;Spinella-Jaegle, et al., 2001). Cell lysates were analyzed for proteincontent using the micro-BCA assay kit (Pierce, Rockford, Ill.). Eachexperiment was done in triplicate.

EXAMPLE 7

Statistical Analyses

Bone disease in multiple myeloma patients was modeled using logisticregression. Independent variables considered were gene expressionintensity values (average difference calls) from ˜10,000 genes (12,625probe sets) measured using version 5.01 MAS (Affymetrix, Santa Clara,Calif.) from 174 cases of newly diagnosed multiple myeloma. The“Signal”, a quantitative measure of gene expression, for each probe setwas transformed to log₂ before entry into the logistic regression modeland permutation-adjustment analysis. There was no prior hypothesis withregard to genes that might be associated with bone disease in myeloma.As a result a univariate model of bone disease for each of the 12,625probe sets was used. Candidate genes were refined using t-tests withpermutation-adjusted significance levels (Westfall and Young, 1993). TheWestfall and Young analysis was used to adjust for the multipleunivariate hypothesis tests. Group differences in DKK1 signal and DKK1protein levels were tested using the Wilcoxon rank sum test. Significantdifferences in patient characteristics by status of bone disease weretested using either the Fisher's exact test or the chi-square test.Expression intensities of genes identified by logistic regression werevisualized with Clusterview (Golub, et. al., 1999). Spearman'scorrelation coefficient was used to measure correlation of geneexpression and protein levels. Significant differences, in osteoblastdifferentiation, between the control and each experimental conditionwere tested using the Wilcoxon rank sum test; separate comparisons weremade for each unique C2C12 experiment. Two-sided p-values less than 0.05were considered significant and two-sided p-values less than 0.10 wereconsidered marginally significant.

EXAMPLE 8

Gene Expression Profiling of Myeloma Cells

To identify genes that were overexpressed and associated with thepresence of bone lesions, comparing microarray data from patients withor without bone lesions were performed. As MRI-defined focal lesions ofbone can occur before radiologically identifiable lytic lesions,T1-weighted and STIR-weighted imaging to evaluate bone lesions wereused. The gene expression patterns of approximately 10,000 genes inpurified plasma cells from the marrow of patients with no bone lesions(n=36) and those with 1 or more (1+) MRI-defined focal lesions (n=137)were modeled by logistic regression analysis. The model identified 57genes that were expressed differently (P<0.0001) in the two groups ofpatients (FIG. 1A). These 57 genes were further analyzed by t-tests withpermutation-adjusted significance (Westfall and Young, 1993). Thesestatistical tests showed that 4 of the 57 genes were overexpressed inpatients with 1+MRI lesions: dihydrofolate reductase (DHFR), proteasomeactivator subunit (PSME2), CDC28 protein kinase 2 (CKS2), and dickkopfhomolog 1 (DKK1). Given that the gene for the Wnt/β-catenin signalingantagonist DKK1 is the only one of the four that codes for a secretedfactor and that Wnt/β-catenin signaling is implicated in bone biology,further tests on DKK1 were carried out. An analysis of the results fromthe 173 patients with myeloma showed that DKK1 signal for patients with1+MRI and no x-ray lesions differ significantly compared to patientswith no MRI and no x-ray lesions (median signal: 2,220 vs. 285; p<0.001)but does not differ significantly compared to patients with 1+MRI and1+x-ray (median signal: 2,220 vs. 1,865; p=0.63) (FIG. 1B, Table 2).

Monoclonal gammopathy of undetermined significance (MGUS) is a plasmacell dyscrasia without lytic bone lesions and can precede multiplemyeloma. In 15 of 16 cases of MGUS, DKK1 was expressed by bone marrowplasma cells at levels comparable to those in multiple myeloma with noMRI or x-ray lesions of bone (FIG. 1B). DKK1 was undetectable in plasmacells from 45 normal donors, and 9 patients with Waldenström'smacroglobulinemia a plasma cell malignancy of the bone lacking bonelesions (FIG. 1B).

TABLE 2 DKK1 mRNA and protein levels in MRI/X-ray-lesion definedsubgroups of MM No MRI/ 1 + MRI/ 1 + MRI/ No X-ray No X-ray 1 + X-ray N36 33 104 DKK1 Mean 536.1 3146.5 3415.1 (Signal) (Std) (720.7) (3079.9)(4870.8) (mRNA) DKK1 Min, 19.2, 16.4, 9.4, 1864.7, (Signal) Median,284.9, 2220.2, 28859.1 (protein) Max 3810.2 10828.4 N 18 9 41 DKK1 Mean9.0 (4.7) 24.0 (17.7) 34.3 (75.3) (ng/ml) (Std) (mRNA) DKK1 Min, 1.8,8.7, 7.4, 20.4, 2.5, 13.5, (ng/ml) Median, 19.7 61.8 475.8 (protein) Max

EXAMPLE 9

Global Gene Expression Reveals DKK-1 and FRZB Linked to Lytic BoneLesion in Multiple Myeloma

In order to further identify the molecular determinants of lytic bonedisease, the expression profiles of ˜12,000 genes in CD138-enrichedplasma cells from newly diagnosed multiple myeloma patients exhibitingno radiological evidence of lytic lesions on bone surveys (n=28) werecompared to those with ≧3 lytic lesions (n=47). The Chi-square test ofabsolute calls (a qualitative measure of gene expression) was used toidentify 30 genes that distinguished the two forms of disease (P<0.05).The Wilcoxon Rank Sum (WRS) test of the signal call (a quantitativemeasure of gene expression) revealed that 104 genes (49 up- and 55down-regulated) differentiated the two disease subtypes (P<0.001).

The Chi-square test identified the RHAMM proto-oncogene as the mostsignificant discriminator between the two groups. It was expressed inonly 7 of 28 patients with no bone disease compared with 34 of 47patients with bone disease (FIG. 2). As expected, plasma cells from only1 of 11 monoclonal gammopathy of undetermined significance expressedRHAMM (FIG. 3). WRS ranked RHAMM as the 14^(th) most significantdiscriminator between the lytic lesion group and no lytic lesion group.NCALD, a calcium binding protein involved in neuronal signaltransduction, was present in 11/28 (40%) of no lytic lesion group butonly in 2/47 (4%) lytic lesion group. Other notable genes identified byChi-square analysis included FRZB, an antagonist of Wnt signaling, thatwas present in 40/47 (85%) of lytic lesion group and 15/28 (53%) of nolytic lesion group. CBFA2/AML1B has been linked to MIP1α expression andwas present in 50% of the no lytic lesion group and in 79% of the lyticlesion group.

PTTG1 (securin) involved in chromosome segregation was identified by WRSas the most significant discriminating gene (P=4×10⁻⁶). It was calledpresent in 11% of no lytic lesion group but present in 50% of the lyticlesion group (FIG. 4). Other notable genes in the WRS test included theTSC-22 homologue DSIPI which was expressed at lower levels in lyticlesion group (P=3×10⁻⁵). DSIPI is also down-regulated in 12 of 12multiple myeloma plasma cells after ex-vivo co-culture with osteoclasts.

In addition, 4 so called “spike genes” were identified that were morefrequently found in lytic lesion group versus no lytic lesion group(p<0.05): IL6, showing spikes in 0/28 no lytic lesion group and 7/47lytic lesion group (p=0.032); Osteonidogen (NID2) showing spikes in 0/28no lytic lesion group and 7/47 lytic lesion group (p=0.032); Regulatorof G protein signaling (RGS13) showing spikes in 1/28 no lytic lesiongroup and 11/47 lytic lesion group (p=0.023); and pyromidinergicreceptor P2Y (P2RY6) showing spikes in 1/28 no lytic lesion group and1/47 lytic lesion group (p=0.023).

Thus, these data suggest that gene expression patterns may be linked tobone disease. In addition to being potentially useful as predictors ofthe emergence of lytic bone disease and conversion from monoclonalgammopathy of undetermined significance to overt multiple myeloma, theymay also identify targets for potential intervention.

EXAMPLE 10

DDK1 and FRZB Tend to be Expressed at Higher Levels in Plasma Cells fromFocal Lesions than from Random Marrow

Given the relationship of DKK-1 and FRZB to lytic lesions, DKK-1 andFRZB expressions were compared in plasma cells derived from random bonemarrow aspirates of the iliac crest with those derived by CT-guided fineneedle aspiration of focal lesions of the spine. These results showedsignificantly higher levels of expression in plasma cells from focallesions.

EXAMPLE 11

DKK-1 and FRZB are Not Expressed in Plasma Cells from Waldenstrom'sMacroglobulinemia

Waldenstrom's macroglobulinemia is a rare plasma cell dyscrasiacharacterized by a monoclonal IgM paraproteinemia and lymphoplasmacyticinfiltration of bone marrow, lymph nodes and spleen. Its clinicalpresentation is quite variable as is the clinical course, yet unlikemultiple myeloma, bone lesions are rare. Although global gene expressionprofiling of CD138-enriched bone marrow plasma cells from 10 cases ofWaldenstrom's Macroglobulinemia reveled gross abnormalities (Zhan etal., 2002), these cells, like normal bone marrow plasma cells, lackexpression of FRZB and DKK (FIG. 20).

EXAMPLE 12

FRZB and Endothelin Receptor B are Correlated with DKK-1

Endothelin 1 is a 21 amino acids vasoconstrictor. Two receptors forendothelin, receptors A and B, have been identified. Breast and prostatecancer cells can produce endothelin 1, and increased concentrations ofendothelin 1 and endothelin receptor A have been found in advancedprostate cancer with bone metastases. Breast cancer cells that producedendothelin 1 caused osteoblastic metastases in female mice. Conditionedmedia and exogenous endothelin 1 stimulated osteoblasts proliferationand new bone formation in mouse calvariae cultures (FIG. 31). Theseresults suggest that endothelin is linked to bone formation.

Table 3 shows that the expression of endothelin receptor B (ENDRB) wascorrelated with that of DKK-1. Endothelin receptor B was a ‘spike’ genein one third of newly diagnosed multiple myeloma (FIG. 29). Endothelinreceptor B was also expressed in subsets of monoclonal gammopathy ofundetermined significance (MGUS) and smoldering multiple myeloma but notin normal plasma cells (FIG. 30).

TABLE 3 Correlation Between Endothelin Receptor B (EDNRB) and DKK-1 GeneSymbol Asymp. Significance (two-tailed) DKK-1 6.35 × 10⁻¹⁴ FRZB 6.59 ×10⁻⁸   EDNRB 0.00014 DKFZP564G202 4.83 × 10⁻¹¹ IFI27 1.43 × 10⁻⁶  SLC13A3 0.00011 CCND1 0.00010 SYN47 4.27 × 10⁻¹⁰ PCDH9 0.00029

EXAMPLE 13

In Vivo Drug Treatment Upregulates DKK-1

It has been shown that DKK-1 expression is massively upregulated by UVirradiation and several other gentoxic stimuli. To see if multiplemyeloma plasma cells also upregulate the genes in response to drugs usedto treat this disease, gene expression profiling of multiple myelomaplasma cells was performed before and after 48 hour in vivo treatmentwith thalidomide (FIG. 33), ImiD (FIG. 34), PS-341 (FIG. 32), ordexamethasone (FIG. 35). These data showed that DKK-1 and FRZBexpression could be massively upregulated in many cases and thussupporting a direct role of DKK-1 in triggering apoptosis of multiplemyeloma plasma cells. It is interesting to note that a newly diagnosedpatient who was primary refractory to all agents tested showed lowlevels of DKK-1 in initial prestudy tests and never showed increasedexpression of DKK-1 or FRZB after drug treatment, supporting a role forDKK-1 expression in promoting apoptosis of multiple myeloma plasmacells. In support of this notion, DKK-1 and FRZB were expressed at lowto undetectable levels in 30 HMCL and several cases of extramedullarydisease (FIG. 15).

EXAMPLE 14

Co-Culture of Multiple Myeloma with Osteoclasts Results in MassiveDownregulation of JUN, FOS, and DKK-1

The close relationship between myeloma cells and osteoclasts isexpressed clinically by the association of debilitating lytic bonedestruction with multiple myeloma. The development of lytic bone lesionsis caused by the activation of osteoclasts through direct and indirectinteractions with myeloma plasma cells. The critical role of osteoclastsin the survival and growth of myeloma cells and in sustaining thedisease process has been gleaned clinically and demonstrated in vivo inexperimental models such as the SCID-hu model for primary human myeloma.

In order to investigate the molecular consequences of multiple myelomaplasma cell/osteoclast interactions, an ex vivo system was developed inwhich CD138-enriched multiple myeloma plasma cells were co-cultured withosteoclasts derived from multiple myeloma peripheral blood stem cells orPBSCs and MNC from healthy donors. CD138-enriched multiple myelomaplasma cells co-cultured with human osteoclasts derived from peripheralblood stem cells from normal donors or multiple myeloma patientsmaintained their viability and proliferative activity as indicated byannexin V flow cytometry, BrdU labeling index and [³H]TdR incorporationfor as long as 50 days. Purity level of plasma cells before and afterco-cultures was greater than 95% as determined by CD38/CD45 flowcytometry.

Microarray analyses of the expression of ˜12,000 genes in 12 multiplemyeloma plasma cells were performed before and after 4 day co-culture.Heirarchical cluster analysis of the 12 multiple myeloma plasma cellspairs and 150 newly diagnosed multiple myeloma plasma cells using 7,913probes sets (genes) revealed that whereas the pre-co-culture sampleswere distributed amongst 3 major cluster groups, the post-co-culturesamples clustered tightly together in 2 of the major branches. Ananalysis of the significant gene expression changes after co-cultureshowed that 95 probe sets (genes) changed 2- to 50-fold (77 up- and 18down-regulated) in at least 8 of the 12 multiple myeloma plasma cellsafter co-culture. CD138-enriched plasma cells from 5 healthy donorsshowed identical shifts in many of the same genes, suggesting thatmultiple myeloma plasma cells do not exhibit altered responses toosteoclasts. However, normal plasma cells as opposed to their malignantcounterparts did not survive in long term co-cultures with osteoclasts.

The most striking changes were in the up-regulation of the chemokinesGRO1, GRO2, GRO3, SCYA2, SCYA8, SCYA18, and IL8. Other notable genesincluded the chemokine receptor CCR1, osteopontin (SPP1), the integrinsITGB2 and ITGB5, matrix metalloproteinase 9 (MMP9), cathepsin K (CTSK)and cathepsin L (CTSL). Surprisingly, a large number ofosteoclast-related genes were among the 77 up-regulated genes. Thedown-regulated genes included cyclin B (CCNB1), the cyclin B specificubiquitin ligase UBE2C, the TSC-22 homologue DSIPI, and JUN, JUND, FOS,and FOSB.

Gene expression changes were also tested in 10 osteoclast cultured aloneand after co-culture with multiple myeloma plasma cells. Twenty-fourgenes (14 up- and 10 down-regulated) changed 2- to 10-fold in at least 7of 10 osteoclasts after co-culture. There were no significantdifferences in gene expression between multiple myeloma plasma cellscultured with osteoclasts derived from multiple myeloma patients or fromhealthy donors, suggesting that multiple myeloma osteoclasts are notqualitatively different than those derived from normal donors.

No significant changes in gene expression were observed when multiplemyeloma plasma cells were cultured in media derived from a co-cultureexperiment, suggesting that contact is important. Given the low ratio ofmultiple myeloma plasma cells to osteoclasts in the co-cultureexperiments (1000:1), it is unlikely that all plasma cells can be incontact with the osteoclasts simultaneously. Thus, it is likely thatsome intercellular communication between multiple myeloma plasma cellsin contact with osteoclasts and those other multiple myeloma plasmacells occurs.

It is known that osteoclasts play a major role in multiple myeloma bonedisease as well as providing multiple myeloma with anti-apoptoticsignals. Recent studies have shown that JUN directly regulates DKK-1expression and that JUN and DKK-1 control apoptosis.

To determine if osteoclasts may prevent apoptosis of multiple myelomaplasma cells by modulating JUN and DKK-1, gene expression profiling wasperformed on purified plasma cells from 12 primary multiple myelomacases before and after 48 hours of co-culture with in vitro derivedosteoclasts. Multiple myeloma plasma cells in the co-culture hadsignificantly higher long-term viability than cells cultured alone. Geneexpression profiling of multiple myeloma plasma cells before and afterosteoclast co-culture revealed that JUN, FOS, and FOSB were 3 of 40genes down-regulated more than 2-fold in all cases (n=12/12).Hierarchical cluster analysis of HMCL and primary multiple myeloma cellswith 95 genes significantly modulated in multiple myeloma plasma cellsafter co-culture revealed a striking similarity between HMCL, primarymultiple myeloma co-cultured with osteoclasts and a subset of newlydiagnosed multiple myeloma in that these cell types had relatively lowlevels of c-JUN and c-FOS.

Importantly, whereas primary multiple myeloma cells show a high degreeof spontaneous apoptosis when cultured alone, multiple myeloma plasmacells cultured in the presence of osteoclasts can survive indefinitely.These data support a link between JUN and DKK-1 and also suggest thatloss of JUN and DKK expression in multiple myeloma may be associatedwith disease progression as extramedulalary diseasse and HMCL, which areinvariably derived from extramedullary disease, lack both JUN and DKK.It is interesting to speculate that one of the major influences ofosteoclasts on multiple myeloma growth and behavior is to downregulateJUN and DKK-1, which directly affects plasma cells apoptosis. Treatmentof HMCL and primary multiple myeloma/osteoclasts co-cultures with DKK-1is expected to result in apoptosis of multiple myeloma plasma cells.DKK-1 will likely have no effect on the osteoclasts, as these cells donot express the Wnt co-receptor LRP-5. Normal bone marrow derived plasmacells also do not express DKK-1 and may help explain their long-livednature.

EXAMPLE 15

Synthesis of DKK1 Protein by Plasma Cells

Serial sections from bone marrow biopsies of 65 cases of multiplemyeloma were stained for the presence of DKK1. The plasma cells in thesecases contained DKK1 in a manner consistent with the gene expressiondata (FIG. 39). Similar experiments with biopsies from 5 normal donorsfailed to identify DKK1 in any cell. There was a strong tendency forDKK1 positive myelomas to have low-grade morphology (abundant cytoplasmwithout apparent nucleoli) with an interstitial growth pattern. Thisstaining was found to be greatest in plasma cells adjacent to bone. DKK1negative myelomas tend to bear high-grade morphology (enlarged nucleiand prominent nucleoli) with a nodular or obliterative growth pattern.In biospies with an interstitial growth pattern, DKK1 was either present(in varying percentages of cells) or absent. In contrast, myelomas withthe more aggressive nodular growth patterns DKK1 was uniformly absent.Importantly, in cases with both interstitial and nodular growth, theinterstitial cells were positive and the nodular cells negative.

EXAMPLE 16

DKK1 Protein in Bone Marrow Plasma

An enzyme-linked immunosorbent assay (ELISA) showed that theconcentration of DKK1 protein in the bone marrow plasma from 107 of the173 newly diagnosed multiple myeloma patients for which gene expressiondata was also available, was 24.02 ng/ml (S.D. 49.58). In contrast, DKK1was 8.9 ng/ml (S.D. 4.2) in 14 normal healthy donors, 7.5 ng/ml (S.D.4.5) in 14 cases of MGUS, and 5.5 ng/ml (S.D. 2.4) in 9 cases ofWaldenström's macroglobulinemia. DKK1 gene expression and the level ofDKK1 in the bone marrow plasma were positively correlated (r=0.65,P<0.001) in the 107 cases of myeloma (FIG. 40A). There was also a strongcorrelation between DKK1 protein levels in bone marrow plasma andperipheral blood plasma in 41 cases of myeloma in which both sampleswere taken simultaneously (r=0.57, P<0.001).

In 68 patients in whom both DKK1 protein levels in the bone marrowplasma and the presence of bone lesions were determined, DKK1 protein inpatients with 1+MRI and no x-ray lesions differ significantly comparedto patients with no MRI and no x-ray lesions (median level: 20 ng/ml vs.9 ng/ml; p=0.002), but does not differ significantly compared topatients with 1+MRI and 1+x-ray lesions (median level: 20 ng/ml vs. 14ng/ml; p=0.36) (FIG. 40B, Table 2).

EXAMPLE 17

Effect of Bone Marrow Serum on Osteoblast Differentiation In Vitro

Bone morphogenic protein-2 can induce differentiation of the uncommittedmesenchymal progenitor cell line C2C12 (Katagiri, et al., 1994) intoosteoblasts through a mechanism that involves Wnt/b-catenin signaling(Bain, et al., 2003; Roman-Roman, et al., 2002). Alkaline phosphatase, aspecific marker of osteoblast differentiation, was undetectable in C2C12cells grown in 5 percent fetal calf serum for 5 days (FIG. 41A).Treatment of C2C12 cells with 50 ng/ml of BMP-2 for 5 days induced themto produce alkaline phosphatase, whereas alkaline phosphatase was notproduced by C2C12 cells that were concomitantly cultured with BMP-2 and50 ng/ml recombinant human DKK1. This in vitro effect on alkalinephosphatase production was neutralized by a polyclonal anti-DKK1antibody, but not by a non-specific polyclonal goat IgG. Bone marrowserum with a DKK1 concentration >12 ng/ml from five patients withmyeloma inhibited the production of alkaline phosphatase by C2C12 cellstreated with BMP-2, and this effect was reversed by the anti-DKK1antibody, but not by non-specific IgG (FIG. 41B). By contrast, C2C12cells treated with 50 ng/ml BMP-2 and 10 percent serum from the bonemarrow of a normal donor induced the production of alkaline phosphataseby the cells (FIG. 41B).

The following references were cited herein:

Zhan et al., Global gene expression profiling of multiple myeloma,monoclonal gammopathy of undetermined significance, and normal bonemarrow plasma cells. Blood 99:1745-1757 (2002).

Zhan et al., Gene expression profiling of human plasma celldifferentiation and classification of multiple myeloma based onsimilarities to distinct stages of late-stage B-cell development. Blood101:1128-1140 (2003).

Fedi et al. Isolation and biochemical characterization of the humanDkk-1 homologue, a novel inhibitor of mammalian Wnt signaling. J BiolChem 274:19465-72 (1999).

Gallea et al. Activation of mitogen-activated protein kinase cascades isinvolved in regulation of bone morphogenetic protein-2-inducedosteoblast differentiation in pluripotent C2C12 cells. Bone 28:491-8(2001).

Spinella-Jaegle et al. Opposite effects of bone morphogenetic protein-2and transforming growth factor-beta1 on osteoblast differentiation. Bone29:323-30 (2001).

Westfall and Young. Resampling-based multiple testing: Examples andmethods for p-value adjustment. Hoboken, N.J.: Wiley-Interscience, 360(1993).

Golub et al. Molecular classification of cancer: class discovery andclass prediction by gene expression monitoring. Science 286:531-7(1999).

Katagiri et al. Bone morphogenetic protein-2 converts thedifferentiation pathway of C2C12 myoblasts into the osteoblast lineage.J Cell Biol 127:1755-66 (1994).

Bain et al. Activated beta-catenin induces osteoblast differentiation ofC3H10T1/2 cells and participates in BMP2 mediated signal transduction.Biochem Biophys Res Commun 301:84-91 (2003).

Roman-Roman et al. Wnt-mediated signalling via LRP5 and beta-catenininduce osteoblast differentiation and mediates the effects of BMP2,American Society of Bone Mineral Research, 2002.

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference.

1. A method of diagnosing lytic bone disease treatable by decreasingDKK-1 expression at the protein level in an individual with multiplemyeloma, comprising: measuring the expression level of the humanhomologue of Dickkopf-1 (DKK-1) protein in said individual, wherein anincreased expression of said protein compared to that in a healthyindividual indicates that said individual has a lytic bone diseasetreatable by decreasing DKK-1 expression at the protein level.
 2. Themethod of claim 1, wherein said expression level is measured byenzyme-linked immunosorbent assay, immunohistochemistry or flowcytometry.
 3. A method of diagnosing DKK1-associated lytic bone diseasein an individual having multiple myeloma, comprising: measuring theexpression level of the human homologue of Dickkopf-1 (DKK-1) protein insaid individual, wherein an increased expression of said proteincompared to that in a healthy individual indicates that said individualhas DKK1-associated lytic bone disease.
 4. The method of claim 3,wherein said expression level is measured by enzyme-linked immunosorbentassay, immunohistochemistry or flow cytometry.